Ethanol (EtOH) consumption results in its metabolism to a toxic intermediate, acetaldehyde (AcH). This reactive metabolite is further metabolized to acetate by enzymes in the Aldh superfamily. During times of heavy alcohol consumption, such as during binge drinking episodes, AcH is not efficiently metabolized to acetate and AcH accumulates, leading to cellular damage and some symptoms of alcohol poisoning. While much of EtOH metabolism occurs in the liver, other cell types are increasingly being shown to metabolize EtOH and exhibit sensitivity to the effects of its metabolism. Stem cells are capable of metabolizing EtOH and many stem cell types express high levels of EtOH-metabolizing enzymes. Because of the carcinogenicity and teratogenicity of EtOH, stem cell metabolism is highly relevant to developmental diseases and cancer. Many of the genetic and bioenergetics circuits that typify adult stem cells are also present in embryonic stem cells (ESCs), making them a relevant model for studying the metabolic consequences of EtOH metabolism in stem cells. However, the molecular mechanisms through which EtOH is metabolized in ESCs remain unclear. Compared to the adult liver, ESCs have different expression profiles of Aldh family members, with Aldh1A1, Aldh1A2, Aldh1B1, and Aldh2 all hypothesized to play roles in EtOH metabolism in stem cells. Preliminary data from our laboratory has shown that Aldh1A2 transcripts are increased by administration of 1 M AcH and Aldh1A1 mRNA levels are decreased by 50 mM and 100 mM EtOH in ESCs. In addition, glycolysis substrates and precursors, and the acylcarnitine transporter SLC22A5 transcripts are increased following treatment of ESCs with EtOH, suggesting changes in energy metabolism. Consequently, we hypothesize that EtOH is metabolized by different Aldh family enzymes in ESCs compared to the adult liver, which leads to different metabolic phenotypes as a result of altered rates of AcH and acetate formation. We will address this hypothesis through a cell culture approach using shRNA technology and untargeted metabolomics. For Aim 1 I will determine which enzymes in the Aldh family metabolize AcH to acetate in ESCs. I will achieve this by knocking down expression of individual Aldh genes using shRNA against Aldh1A1, Aldh1A2, Aldh1A3, Aldh1B1, and Aldh2, and determining the ability of these enzymes to metabolize AcH to acetate once EtOH is added to the cells. In Aim 2 I will determine the metabolic signatures of ESCs following the addition of EtOH using an untargeted metabolomics approach. This will provide a high-throughput approach for identifying novel metabolites, cofactors, and pathways that contribute to EtOH actions in ESCs. Inhibition of Aldh activity using the Aldh inhibitor diethylaminobenzaldehyde and by specific shRNA knockdown against individual Aldh family members shown to metabolize AcH will reveal pathways altered by the production of AcH from EtOH in ESCs. Completion of this project will increase our understanding of how stem cells metabolize EtOH and may provide insight into alleviation of EtOH-induced complications through dietary or pharmacological interventions.